The goal of this work is to develop salivary biomarkers for chronic Graft Versus Host Disease (cGVHD). This will be done using an iTRAQ marker-based, quantitative mass spectrometry (MS) approach to detect cGVHD-associated protein expression changes in resting whole saliva samples from patients who received hematopoietic cell transplants (HCT) and subsequently developed cGVHD. Saliva is proposed for this study not simply because it is convenient to obtain, but primarily because salivary glands are a lymphocyte-homing target in the mucosal immune system, where we postulate GVHD is activated (Murai et al., Nat Immunol 4:154-160, 2003). Hence, salivary protein composition changes should occur early in the disease and should correlate with disease intensity. The etiology of GVHD predicts increases in salivary inflammatory cytokine and related cell signaling protein contents. Our study will consist of discovery and confirmation/validation phases. In the discovery phase, an iTRAQ-based quantitative MALDI TOF/TOF mass spectrometry technique will be used to identify potential candidate cytokine/chemokine and other protein biomarkers for cGVHD in pooled samples of resting whole saliva from [HCT recipients with or without cGVHD.] In the validation phase, the eight candidate biomarker proteins showing the greatest cGVHD-associated expression differences will be measured using [ELISA] assays to confirm the MS results in the pooled samples, and to establish expression difference magnitudes and prevalence in the individual patient samples. The protein expression changes in the individual saliva samples will be used to construct a biomarker panel of 5 proteins that will be evaluated for an ability to predict cGVHD. The MS analyses will be directed by our collaborator, Dr. Jing Zhang, Shaw Professor of Neuropathology at the UW, who has developed MS techniques for identifying CSF biomarkers for central nervous system diseases. We propose to apply his techniques to find salivary biomarkers for cGVHD. We have preliminary results showing control whole saliva can be analyzed by the proposed MS procedures. The proposed state-of-art MS proteomics approach should yield specific GVHD biomarkers that would allow more timely detection of GVHD, leading to earlier treatment, and consequently less disease severity in the oral and other target tissues of cGVHD. [Since many disease biomarkers arise from underlying pathophysiologic mechanisms, function-analysis of the array of biomarkers found here should give insight into the pathogenesis of cGVHD.] Graft Versus Host Disease (GVHD) is a frequent, serious, potentially life-threatening complication of hematopoietic cell transplants used to treat hematologic disorders as well as other diseases. GVHD can occur as acute or chronic disease, and it is currently difficult to predict who will develop extensive disease. The proposed etiology of the disease suggests salivary glands should have an early involvement in GVHD, and salivary protein changes could allow early detection and treatment, which could decrease the seriousness and mortality of the disease, [as well as give insight into disease etiology]. The goal of this proposal is to develop such a salivary assay. [unreadable] [unreadable] [unreadable]